Project Summary Neuroblastoma (NB) is a cancer of the developing sympathetic nervous system that most commonly affects young children. Despite intense multi-modal therapy, the overall survival rate for high-risk NB remains less than 50%, and relapsed NB is almost universally incurable. Our long-term goal is to improve the outcome of children with NB by defining the inherited and acquired genetic/genomic basis of the disease and response to therapy. Structural variations (SVs) are changes in chromosomal architecture such as deletions, duplications, translocations, and inversions. SVs can be inherited in the germline or acquired somatically, and have been implicated in a wide array of human diseases, including cancer. While array-based approaches can identify large scale copy number variation (CNV), whole-genome sequencing (WGS) enables characterization of many complex SVs at base-pair resolution. Our objective here is to identify SVs associated with NB susceptibility, malignant progression, and clonal evolution mediating therapy resistance that may be exploitable for risk prediction and/or therapeutically. Our central hypothesis is that germline and somatic SVs potently influence tumorigenesis and acquisition of therapy resistance in NB. The motivation for the proposed work is the need for an integrated understanding of inherited genetic variation and acquired mutational events in the host and tumor to enable improved risk assessment and develop rational, evidence-based therapies to improve patient outcomes. We will test our hypothesis in three specific aims: 1) Identify rare germline SVs associated with NB through a SV-based association study of 7,500 NB cases and 15,000 healthy children as controls and evaluation of heritability in 642 patient-parent trios. 2) Discover and prioritize somatic SVs using whole genome-sequencing (WGS) of 146 diagnostic NB pairs (tumor, germline), and 60 relapsed NB trios (diagnostic, relapsed and germline). Recurrent and/or clonally enriched SVs will be prioritized using an integrative transcriptomic and (epi)genomic approach and validated in two independent cohorts comprised of 1,158 NBs. 3) Characterize and determine how genes influenced by recurrent SVs in NB drive tumorigenesis and the malignant phenotype. For the final aim, a two-pronged approach is planned: 3a) a detailed in silico molecular characterization of SVs and genes transcriptionally altered by SVs using matched germline and tumor data from 646 primary patient samples (germline/tumor whole genome sequencing, DNA copy number, DNA methylation, and mRNA/miRNA expression) and 3b) genetic manipulation of candidate genes and/or genome-editing of prioritized SV loci in a highly characterized panel of NB cell lines to define biological significance and begin to understand underlying mechanisms of tumorigenesis. This work will have a sustained and positive impact on the field by providing substantial insights into genetic predisposition, malignant progression, and the relapsed genome of this important childhood cancer. Ultimately, this knowledge has the potential to inform the development of clinical biomarkers and/or novel, evidence-based therapies to improve outcomes of children with NB, and possibly other cancers.